The small-subunit processome is a ribosome assembly intermediate

Abstract

The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5' end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.

FIG. 1.

Schematic diagram of pre-rRNA processing in S. cerevisiae. The 35S pre-rRNA is transcribed as a single transcript that is subsequently cleaved at the A₀, A₁, and A₂ sites by the SSU processome. Cleavage at A₂ or A₃ separates precursors to the small-subunit (18S) and large-subunit (5.8S and 25S) rRNAs, whereas cleavage at A₀ or A₁ matures the 5′ end of the 18S rRNA. The broken line represents multiple processing steps. The locations of oligonucleotides a, b, c, e, and y, used to probe for precursor and mature rRNAs in subsequent experiments, are shown.

FIG. 2.

A subset of ribosomal proteins is found in the SSU processome. 3×HA-tagged ribosomal proteins Rps4, Rps6, Rps7, Rps9, Rps14, Rps27, Rps28, and Rpl33 were immunoprecipitated with beads conjugated with HA antibodies. 3×HA-tagged Utp9 (a bona fide SSU processome component) and YPH499 (the untagged parental strain) were used as positive and negative controls, respectively. Ribosomal proteins were tested with Western blotting for their abilities to coimmunoprecipitate Mpp10. Ribosomal proteins were also tested with Northern blotting for their abilities to coimmunoprecipitate the U3 snoRNA. Results for totals (lanes T), representing 5% (Western blot) and 10% (Northern blot) of the proteins extracted, and immunoprecipitates (lanes IP) are shown.

FIG. 3.

Subcellular localization of new SSU processome proteins and two other related proteins. All proteins were 3×HA tagged and colocalized using anti-HA(red) or nucleolar protein Mpp10 (green) with anti-Mpp10 by indirect immunofluorescence. DAPI (4′,6′-diamidino-2-phenylindole) (blue) was used to stain the nuclear DNA. Merge, merge of tagged protein, Mpp10, and DAPI. (A) Nucleolar localization of Utp18, Noc4, Utp20, Utp21, Utp22, and Emg1. The parental strain YPH499 (untagged) and Utp4 (a bona fide SSU processome component) were used as negative and positive controls, respectively. (B) Nucleolar localization of Bfr2 and Enp2.

FIG. 4.

Coimmunoprecipitation experiments define new proteins as SSU processome components. Immunoprecipitations of 3×HA-tagged Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, Krr1, and Enp1 were carried out using anti-HA antibodies. 3×HA-tagged proteins were immunoprecipitated and tested for their ability to coimmunoprecipitate with Mpp10 as determined by Western blot analysis. U3 snoRNA that coimmunoprecipitated with 3×HA-tagged proteins were analyzed by Northern blotting. Results for totals (lanes T), representing 5% (Mpp10) or 10% (U3 snoRNA) of the total protein extracted, and for immunoprecipitates (lanes IP) are shown. The parental strain, YPH499 (untagged), and 3×HA-tagged Rpf2 (a protein involved in large-subunit biogenesis) (40) were used as negative controls. 3×HA-tagged Utp7 (a bona fide SSU processome component) was used as a positive control.

FIG. 5.

Depletion of new SSU processome proteins leads to defects in pre-18S rRNA processing. Strains expressing Utp1, Utp7, Utp18, Noc4, Utp20, Emg1, Bfr2, and Enp1 from galactose-inducible and glucose-repressible promoters were grown in early log phase (time point 0) in galactose- and raffinose-supplemented media (undepleted) and then shifted into glucose (depleted) for 24 h. YPH499, the untagged parental strain, was used as a control. RNA from undepleted or depleted yeast strains was analyzed for the presence of pre-rRNAs by Northern blotting. Equal amounts of RNA were separated on formaldehyde-1.2% agarose gels, transferred to membranes, and hybridized with the specific oligonucleotide probes shown in Fig. 1. (A) Northern blot using oligonucleotide c, which hybridizes to 35S, 32S, 27SA₂, 23S and 21S pre-rRNAs; (B) Northern blot using oligonucleotides b and e, which hybridize to the 35S, 32S, 27SA, 27SB, 23S, 20S pre-rRNAs; (C) Northern blot using oligonucleotides a and y, which hybridize to the 18S and 25S rRNAs, respectively.

FIG. 6.

SSU processome and small-ribosomal-subunit proteins associate with precursor rRNAs. 3×HA-tagged Imp4, Utp1, Utp4, Utp9, Utp16, Utp17, Utp20, Utp21, Rps4, Rps6, and Rpf2 were immunoprecipitated with anti-HA antibodies bound to Sepharose beads and analyzed by Northern blotting for their ability to coimmunoprecipitate with pre-rRNAs (lanes HA). An untagged strain, YPH499, and 3×HA-tagged Rpf2, a protein involved in large-subunit biogenesis, were used as controls. Imp4, a known SSU processome component, was used as a positive control. All experiments were also done in parallel without antibody, i.e., with beads alone (lanes BA), and pre-rRNA precursors that immunoprecipitated were determined by comparison to a standard (lane STD). Northern blots were probed with oligonucleotides b, c, and e, which hybridize to the 35S, 32S, 27SA2, 27SB, 23S, 21S, and 20S pre-rRNAs (as shown in Fig. 1). Rps4 and Rps6 Northern blots were probed with oligonucleotides b and e, which hybridize to the 35S, 32S, 23S, 21S, and 20S pre-rRNAs (see Fig. 1).